US8476490B2 - Method for production of moth orchid having modified flower color - Google Patents

Method for production of moth orchid having modified flower color Download PDF

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US8476490B2
US8476490B2 US12/596,739 US59673908A US8476490B2 US 8476490 B2 US8476490 B2 US 8476490B2 US 59673908 A US59673908 A US 59673908A US 8476490 B2 US8476490 B2 US 8476490B2
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moth orchid
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Satoshi Araki
Takanori Suzuki
Kouichi Katayama
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Ishihara Sangyo Kaisha Ltd
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)

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  • the present invention relates to a method for modify the flower color of a moth orchid by employing gene recombination technology. Particularly, it relates to a method for producing a moth orchid having a red flower color by introducing genes encoding enzymes related to biosynthesis of cyanidin into a moth orchid.
  • the color of flower is a particularly important character in ornamental plants, and flowers having various colors have been produced by cross breeding heretofore.
  • a specific character such as flower color
  • a period of cross breeding varies depending on plant species, and in the cases of orchids such as moth orchids, which take a long time for blossom, it is practically impossible to produce a variety with a desired character only by cross breeding. Therefore, despite the demand of the market, it has been extremely difficult to change the flower color of a moth orchid to a desired color.
  • the color of flower derives mainly from three types of pigments: anthocyanin, carotenoid and betalain.
  • anthocyanin is the pigment having the broadest maximum absorption wavelength range and governs colors from red to blue.
  • Anthocyanin is a kind of flavonoid and biologically synthesized through a metabolic pathway shown in FIG. 1 .
  • the color of anthocyanin substantially depends on its chemical structure, especially on the number of hydroxyl groups on the B ring.
  • the hydroxylation of the B ring is catalyzed by a flavonoid 3′-hydroxylase (F3′H) and a flavonoid 3′,5′-hydroxylase (F3′5′H).
  • F3′H activity When there is neither F3′H activity nor F3′5′H activity in petal cells, pelargonidin (orange) is synthesized, and when there is F3′H activity, cyanidin (red) is synthesized. Further, when there is F3′5′H activity, delphinidin (blue) is synthesized. Therefore, in order to produce the red flower color, the role of F3′H is considered to be important.
  • Patent Document 1 In a case of genetic alteration of flower color, a blue rose was produced using a F3′5′H gene from viola ⁇ wittrockiana (Patent Document 1).
  • Non-Patent Document 1 alteration of the flower color of a moth orchid from pink to magenta by overexpressing an endogenous gene was reported.
  • Patent Document 1 WO2005/017147
  • Non-Patent Document 1 Su and Hsu, Biotechnology Letters (2003) 25: 1933-1939.
  • the present inventors have conducted an extensive study on introduction of genes encoding enzymes related to biosynthesis of cyanidin, and as a result, they found that introduction of the genes encoding four enzymes downstream of the naringenin into petal cells of a white moth orchid enables the petal cells to synthesize the red pigment cyanidin. Thus, the present invention has been accomplished.
  • the present invention is based on the discovery and provides:
  • a method for producing a moth orchid having a modified flower color which comprises transfecting a moth orchid with a gene encoding a flavanone 3-hydroxylase, a gene encoding a flavonoid 3′-hydroxylase, a gene encoding a dihydroflavonol 4-reductase and a gene encoding an anthocyanidin synthase and expressing the genes.
  • “flavanone 3-hydroxylase” (hereinafter referred to also as F3H) is an enzyme which catalyzes a reaction which produces dihydrokaempferol from naringenin.
  • “Flavonoid 3′-hydroxylase” (hereinafter referred to also as F3′H) is an enzyme which catalyzes a reaction which produces dihydroquercetin from dihydrokaempferol.
  • DFR Dihydroflavonol 4-reductase
  • ANS antagonisthocyanidin synthase
  • FIG. 1 is a synthetic pathway of anthocyanin.
  • FIG. 2 is a plasmid vector used for transfecting a petal with a gene.
  • P-CaMV35S is a cauliflower mosaic virus 35S promoter.
  • ⁇ is an omega sequence of tobacco mosaic virus.
  • T is a transcription termination sequence derived from a cauliflower mosaic virus.
  • FIG. 3 shows identification of the genes necessary for changing the petal color of a white moth orchid to red.
  • FIG. 4 shows transfection of a petal of Phal. amabilis with F3′H genes derived from different plants.
  • FIG. 5 shows transfection of a petal of Phal. amabilis with DFR genes derived from different plants.
  • FIG. 6 shows transfection of a petal of Phal. amabilis with ANS genes derived from different plants.
  • FIG. 7 is a schematic view showing a procedure for construction of DNA for transformation of moth orchid.
  • FIG. 8 is a schematic view showing a procedure for construction of DNA for transformation of moth orchid.
  • the genes encoding F3H, F3′H, DFR and ANS novel or conventional genes may be used.
  • the gene encoding F3H may be a gene registered on GenBank (accession number: DQ394303, AY221246, AJ493133, AY641730, AF184270, AB078956, AB201760, AY669324, AF036093, AB211958, AB187027 or AB234905)
  • the gene encoding F3′H may be a gene registered on GenBank (accession number: AAG49298, ABA64468, BAE47004, BAE47005, BAE47006, BAE71221, BAE72874, CAI54278, NP — 920627 or Z17221)
  • the gene encoding DFR may be a gene registered on GenBank (accession number: AB012924, AAB62873, AAC17843, AAD49343, AAQ83576, A
  • novel genes which were found in the present invention such as the gene encoding F3H (SEQ ID NO: 9), the gene encoding F3′H (SEQ ID NO: 1), the gene encoding DFR (SEQ ID NO: 15) and the gene encoding ANS (SEQ ID NO: 11) from a moth orchid may also be used.
  • the gene encoding F3′H has a DNA sequence represented by SEQ ID NO: 3
  • the gene encoding DFR has a DNA sequence represented by SEQ ID NO: 7
  • the gene encoding ANS has a DNA sequence represented by SEQ ID NO: 13.
  • the gene encoding DFR has a DNA sequence represented by SEQ ID NO: 5.
  • the gene encoding F3H has a DNA sequence represented by SEQ ID NO: 9
  • the gene encoding F3′H has a DNA sequence represented by SEQ ID NO: 1
  • the gene encoding ANS has a DNA sequence represented by SEQ ID NO: 11.
  • the gene encoding F3′H it is preferred to use the gene derived from a moth orchid or Gerbera , especially the gene derived from a moth orchid.
  • the gene encoding DFR it is preferred to use the gene derived from Gerbera or Torenia , especially the gene derived from Torenia .
  • the gene encoding a flavonoid 3′-hydroxylase (F3′H) is the gene derived from a moth orchid or Gerbera
  • the gene encoding a dihydroflavonol 4-reductase (DFR) is the gene derived from Gerbera or Torenia , because the flower color would be deep red.
  • the term, gene is used in the broadest sense and means an isolated nucleic acid molecule having a DNA sequence encoding the amino acid sequence of the enzyme or a DNA sequence complementary thereto.
  • the nucleic acid molecule may contain, in addition to the exon encoding the amino acid sequence, an intron, a 5′ untranslated region and a 3′ untranslated region.
  • a gene derived from a specific plant means a gene registered on GenBank as derived from the plant.
  • the gene may be a spontaneously or artificially mutated gene having a high similarity to the registered gene.
  • the high similarity means that the homology between the amino acid sequences encoded by them is at least 85%, preferably at least 90%, more preferably at least 95%, further preferably at least 98%.
  • genes are introduced into a moth orchid through a recombinant vector, especially an expression vector.
  • the expression vector has an expression regulation region such as a promoter, a terminator, a DNA replication origin, etc. suitable for the host species into which the genes are introduced.
  • the promoter may be a promoter which induces gene expression in petal cells, such as the CHS gene promoter in moth orchid or the cauliflower mosaic virus (CaMV) 35S promoter.
  • the terminator may, for example, be the CHS gene terminator in moth orchid or the cauliflower mosaic virus (CaMV) 35S terminator.
  • the expression vector can be produced in accordance with a conventional method by using a restriction endonuclease, a ligase, etc. Further, transformation of hosts with the expression vector can also be carried out in accordance with a conventional method.
  • microprojectile bombardment Agrobacterium -mediated transformation, electroporation, PEG-mediated transformation or virus-mediated transformation may, for example, be used.
  • FIG. 7 and FIG. 8 show examples of the vectors used for Agrobacterium -mediated transformation of a moth orchid.
  • Gene expression is used in the broadest sense and may involve RNA production only or both RNA production and protein production. Gene expression in a plant may be constitutive, developmental or inducible, and may be systemic or tissue-specific.
  • plants to be transformed are Orchids, preferably moth orchids.
  • Moth orchids include those of the genus Phalaenopsis and those of the genus Doritaenopsis .
  • the present invention relates to a method for producing a moth orchid having a red flower color by changing the flower color of a moth orchid by introducing a gene encoding F3H, a gene encoding F3′H, a gene encoding DFR and a gene encoding ANS into the moth orchid and expressing the genes.
  • a gene encoding F3H a gene encoding F3′H
  • a gene encoding DFR a gene encoding ANS into the moth orchid and expressing the genes.
  • the gene encoding F3′H is derived from a moth orchid or Gerbera;
  • the gene encoding F3′H has a DNA sequence represented by SEQ ID NO: 1 or 3;
  • the gene encoding DFR has a DNA sequence represented by SEQ ID NO: 5 or 7;
  • the gene encoding F3′H is derived from a moth orchid or Gerbera
  • the gene encoding DFR is derived from Gerbera or Torenia
  • gene encoding F3′H has a DNA sequence represented by SEQ ID NO: 1 or 3
  • gene encoding DFR has a DNA sequence represented by SEQ ID NO: 5 or 7;
  • the gene encoding F3H has a DNA sequence represented by SEQ ID NO: 9
  • the gene encoding F3′H has a DNA sequence represented by SEQ ID NO: 1 or 3
  • the gene encoding DFR has a DNA sequence represented by SEQ ID NO: 5 or 7
  • the gene encoding ANS has a DNA sequence represented by SEQ ID NO: 11 or 13.
  • homology searching program for examining an amino acid sequence encoded by the novel gene of the present invention and known amino acid sequences
  • BLASTP 2.2.15 http://www.ddbj.nig.ac.jp/search/blast-j.html, Document: Altschul et al., Nucleic Acids Res. (1997) 25: 3389-3402) was used.
  • homology is the degree of similarity of amino acids over the entire sequence, and its value is obtained by aligning an amino acid sequence encoded by the novel gene and known amino acid sequences in order of the high similarity, and dividing the number of the homologous amino acids between them by the number of amino acids of the compared region.
  • high similarity is an alignment result output under a state (with gap, expected value 10, with filter) that the above BLASTP program parameter was defaulted.
  • the homology described in the present Examples is one described with respect to a known amino acid sequence which showed the highest homology in the above homology analysis.
  • petals of moth orchid were transfected with the various genes by the following gene transfection method, and their property was evaluated. All genes had a DNA structure having a promoter at 5′ side and a terminator at 3′ side and were introduced into petal cells in a form expressible in the cells.
  • a bud of moth orchid was sterilized with a 1 wt % of sodium hypochlorite aqueous solution for five minutes and washed with sterilized water 3 times. Then, the bud was resolved into a lateral sepal, dorsal sepal and petal, and the lateral sepal, dorsal sepal and petal were left on an agar medium containing NDM salt (Tokuhara and Mii, Plant Cell Reports (1993) 13: 7-11.) and 0.6 wt % agarose.
  • NDM salt Yamahara and Mii, Plant Cell Reports (1993) 13: 7-11.
  • a bud having a length of about 15 mm of a white moth orchid Phal. amabilis was used.
  • DNA to be introduced was purified by using Hi Speed Plasmid Midi Kit (QIAGEN), and genes were introduced by microprojectile bombardment. Further, when plural genes were introduced simultaneously, an equal mixture of solutions of DNAs of these genes was used as a DNA solution for transfection. DNA was adsorbed onto gold particles in the following ratio.
  • a DNA solution in 20 ⁇ l of TE buffer (containing 2 ⁇ g plasmid DNA carrying a gene) was mixed with 50 ⁇ l of a gold particle suspension (the particle size: 1.0 ⁇ m, 60 mg/ml in 50% glycerol), and 50 ⁇ l of 2.5 M calcium chloride and 10 ⁇ l of 0.2 M spermidine were added to 70 ⁇ l of the mixture and suspended, to allow adsorption of DNA on the gold particles. After centrifugation, the supernatant was removed, and the particles were washed with 70% ethanol and 100% ethanol respectively. Then, the precipitate was suspended in 60 ⁇ l of 100% ethanol. The suspension was used as a sample solution, and 10 ⁇ l of the sample solution was used for one gene transfection.
  • a gold particle suspension the particle size: 1.0 ⁇ m, 60 mg/ml in 50% glycerol
  • 50 ⁇ l of 2.5 M calcium chloride and 10 ⁇ l of 0.2 M spermidine were added to 70 ⁇ l of the
  • IDERA GIE-III TANAKA Co., Ltd.
  • the gene transfection was carried out with a distance from a nozzle to a sample of 12 cm, under a reduced pressure of ⁇ 80 kPa, a helium gas pressure of 0.3 MPa with a spraying time of 0.025 second.
  • petals were left on an NDM salt agar medium and cultured under a light-dark cycle (light intensity: 23 ⁇ mol/m 2 /s, light period: 16 hours, dark period: 8 hours) at 25° C.
  • pBS-P35T35 is a plasmid having the cauliflower mosaic virus 35S promoter (Hohn et al., Curent Topics in Microbiology and Immunology (1982) 96: 194-236), an omega sequence of tobacco mosaic virus (Gallie et al., Nucleic Acids Research (1987) 15: 3257-3273), a restriction endonuclease SwaI site and the cauliflower mosaic virus 35S terminator in this order in pBluescriptIISK—(Stratagene) ( FIG. 2 ).
  • a plasmid having substantially the same function as pBS-P35T35 can be constructed as follows.
  • oligonucleotide SAS-S (5′-CTAGCTAGCGGCGCGCCTGCAGGATATCATTTAAATCCCGGG-3′; SEQ ID NO: 17) and an oligonucleotide SAS-AS (5′-CCCGGGATTTAAATGATATCCTGCAGGCGCGCCGCTAGCTAG-3′; SEQ ID NO: 18) were thermally denatured, then gradually cooled to room temperature and treated with NheI, and the resulting fragment was ligated between the XbaI-EcoRV sites of pBluescriptIISK—(Stratagene) to prepare a plasmid DNA with a modified restriction site, pBS-SAS.
  • PCR was carried out by using a cauliflower mosaic virus genome DNA (GenBank accession V00140) as the template, a primer T-CaMV35S-Ssel-F (5′-AACCTGCAGGAAATCACCAGTCTCTCTCTA-3′; SEQ ID NO: 19) and a primer T-CaMV35S-AscI-R (5′-GGCGCGCCATCGATAAGGGGTTATTAG-3′; SEQ ID NO: 20), and the amplified region was treated with restriction endonucleases Sse83871 and AscI. The resulting fragment was ligated between the Sse8387′-AscI sites of pBS-SAS to prepare pBS-T35S.
  • a cauliflower mosaic virus genome DNA GenBank accession V00140
  • the cauliflower mosaic virus 35S promoter and the tobacco mosaic virus omega sequence were cut out from pJD301 (Leon et al., Plant Physiology (1991) 95: 968-972) with HindIII and HincII and ligated between the HindIII-SmaI sites of pBS-T35S to prepare an expression vector (pBS-P35T35).
  • PhCHS3 F1 (5′-AAGCTTGTGAGAGACGACGGA-3′; SEQ ID NO: 21) and PhCHS3 R1 (5′-TGGCCCTAATCCTTCAAATT-3′; SEQ ID NO: 22) designed from a known moth orchid CHS gene (PhCHS) sequence (GenBank accession No.: DQ089652) were used as the primers.
  • the reaction was carried out by repeating a step of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 1 minute 25 cycles. Amplification was carried out again in the same reaction solution under the same conditions again using the reaction product as the template.
  • p35PhCHS3 is DNA for expressing the moth orchid CHS gene in plant cells.
  • CHI genes from various plants have been reported (GenBank accession Nos.: AY700850, AY086088, DQ160231, AJ004902, AF474923, XM — 470129, U03433 and AB187026), in the PCR, CHI-dgF1 (5′-TTYCTCGSYGGBGCMGGYGWVMGVGG-3′; SEQ ID NO: 25) and CHI-dgR1 (5′-CMGGIGAIACVSCRTKYTYICCRATVAT-3′; SEQ ID NO: 26) designed from the conventionally known CHI gene were used as the primers. In the reaction, a step of 94° C. for 30 seconds and 72° C.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer CHI-dgF3 (5′-TMIKYWCMGGISMITTYGARAARYT-3′; SEQ ID NO: 27) and a primer CHI-dgR3 (5′-TYICCRATVATIGWHTCCARIAYBGC-3′; SEQ ID NO: 28).
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) to obtain PhCHIfrag 16, and a partial DNA sequence of PhCHIfrag 16 was determined (PhCHI partial sequence).
  • the 3′ sequence and the 5′ sequence were analyzed by RACE.
  • 3′ RACE was carried out by using a primer designed from the PhCHI partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhCHI-GSP F1 (5′-ATGCTGCTGCCATTAACGGGTCA-3′; SEQ ID NO: 29) and GeneRacer 3′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • PhCHI-GSP F2 5′-TCCGAGAAGGTCTCCGGGAACT-3′; SEQ ID NO: 30
  • GeneRacer 3′ Nested primer 5′-TCCGAGAAGGTCTCCGGGAACT-3′; SEQ ID NO: 30
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) to obtain PhCHI3′RACE23.
  • the 3′ DNA sequence of PhCHI3′RACE23 was determined (PhCHI 3′RACE sequence).
  • 5′RACE was carried out by using a primer designed from the PhCHI partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhCHI-GSP R1 (5′-GCATTCGTCAGCTTCTTGCTCTCT-3′; SEQ ID NO: 31) and GeneRacer 5′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer PhCHI-GSP R2 (5′-ATCACATCAGTCTCAGCCACA-3′; SEQ ID NO: 32) and GeneRacer 5′ Nested primer.
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) to obtain PhCHI5′RACE54.
  • the 5′ DNA sequence of PhCHI5′RACE54 was determined (PhCHI5′RACE sequence).
  • the full length of the moth orchid CHI gene was cloned on the basis of the PhCHI3′RACE sequence and the PhCHI5′RACE sequence.
  • PCR was carried out by using the above-mentioned cDNA, a primer PhCHI init (5′-ATGGCAGAAACAGTGGCGACGCCCA-3′; SEQ ID NO: 33) and a primer PhCHI term (5′-TCAAACGACTCCATCTTGCTC-3′; SEQ ID NO: 34).
  • a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 45 cycles.
  • p35PhCHI1 DNA sequence of the full-length moth orchid CHI gene in p35PhCHI1 was determined (PhCHI1, SEQ ID NO: 35), and the gene having the sequence from moth orchid was found to be novel. Homology analysis indicated that the amino acid sequence encoded by the DNA sequence has 54% homology to the amino acid sequence (GenBank accession No.: DQ120521) encoded by the CHI gene of tea plant.
  • p35PhCHI1 is DNA for expressing the moth orchid CHI gene in plant cells.
  • F3H genes from various plants have been reported (GenBank accession Nos.: DQ394303, AY221246, AJ493133, AY641730, AF184270, AB078956, AB201760, AY669324, AF036093, AB211958, AB187027 and AB234905), in the PCR, a primer F3H-dgF1 (5′-TIVGIGAYGARGABGARMGBCCIAA-3′; SEQ ID NO: 37) and a primer F3H-dgR1 (5′-ACBGCYYGRTGRTCHGCRTTCTTRAA-3′; SEQ ID NO: 38) designed from the conventionally known F3H gene were used as the primers.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, and 70° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer F3H-dgF3 (5′-AARYTBRGKTTYGAYATGWCHGGIG-3′; SEQ ID NO: 39) and a primer F3H-dgR3 (5′-GGHWSRACVGTDATCCAIGWBTT-3′; SEQ ID NO:40).
  • the 3′ sequence and the 5′ sequence were analyzed by RACE.
  • 3′ RACE was carried out by using a primer designed from the PhF3H partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhF3H-GSPF1 (5′-TTCTCATACCCAATCGGGAG-3′; SEQ ID NO: 41) and GeneRacer 3′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • PhF3H-GSP F2 5′-AATCGGGAGCCGCGATTACT-3′; SEQ ID NO: 42
  • GeneRacer 3′ Nested primer 5′-AATCGGGAGCCGCGATTACT-3′; SEQ ID NO: 42
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) to obtain PhF3H3′RACE33.
  • the 3′ DNA sequence of PhF3H3′RACE33 was determined (PhF3H 3′RACE sequence).
  • 5′RACE was carried out by using a primer designed from the PhF3H partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhF3H-GSPR1 (5′-TCTGTGTGGCGCTTCAGGCC-3′; SEQ ID NO: 43) and GeneRacer 5′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution, PhF3H-GSP R2 (5′-TGAGGTCCGGTTGCGGGCATTTT-3′; SEQ ID NO: 44) and GeneRacer 5′ Nested primer.
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) to obtain PhF3H5′RACE86.
  • the 5′ DNA sequence of PhF3H5′RACE86 was determined (PhF3H 5′RACE sequence).
  • the full length of the moth orchid F3H gene was cloned on the basis of the PhF3H 3′RACE sequence and the PhF3H 5′RACE sequence.
  • PCR was carried out by using the above-mentioned cDNA, a primer PhF3H init. (5′-ATGGCCCCAATACCATTCCTACCGA-3′; SEQ ID NO: 45) and a primer PhF3H term. (5′-CCTTAAGCTAAAATCTCATTTAATGCCTTTGCTCC-3′; SEQ ID NO: 46).
  • a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 40 cycles.
  • p35PhF3H1 DNA sequence of the full-length moth orchid F3H gene in p35PhF3H1 was determined (PhF3H1; SEQ ID NO: 9), and the gene having the sequence from moth orchid was found to be novel. Homology analysis indicated that the amino acid sequence encoded by the DNA sequence has 86% homology to the amino acid sequence (GenBank accession No.: X89199) encoded by the F3H gene of Bromheadia finlaysoniana .
  • p35PhF3H1 is DNA for expressing the moth orchid F3H gene in plant cells.
  • F3′H genes from various plants have been reported (GenBank accession Nos.: AAG49298, ABA64468, BAE47004, BAE47005, BAE47006, BAE71221, BAE72874, CAI54278 and NP — 920627), in the PCR, a primer F3HDF3 (5′-GCITAYAAYTAYCARGAYYTIGTNTT-3′; SEQ ID NO: 47) and a primer F3HD-R4-2 (5′-GCICKYTGIARIGTNARNCC-3′; SEQ ID NO: 48) designed from the above-mentioned F3′H genes were used as the primers. In the reaction, a step of 94° C. for 30 seconds, 48° C.
  • Nested PCR was carried out by using the resulting reaction solution, a primer F3HDF4 (5′-GAYYTIGTNTTYGCICCNTAYGG-3′; SEQ ID NO: 49) and a primer F3HD-R3-2 (5′-DATICKICKICCIGCNCCRAANGG-3′; SEQ ID NO: 50).
  • a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 1 minute was repeated 35 cycles.
  • Nested PCR was carried out again by using the resulting reaction solution, a primer F3HDF5 (5′-GARTTYAARIIIATGGTIGTIGARYTNATG-3′; SEQ ID NO: 51) and a primer F3HD-R1-3 (5′-GGRTCICKNGCDATNGCCC-3′; SEQ ID NO: 52).
  • a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 30 seconds was repeated 35 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) and designated as PhF3′H-D/pCR4.
  • PhF3′H partial sequence A partial DNA sequence of PhF3′H-D/pCR4 was determined (PhF3′H partial sequence).
  • 3′ RACE was carried out by using a primer designed from the PhF3′H partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhF3dH-F7 (5′-AGGGCGAAGTTAATGGTGGAGGCAGTGATA-3′; SEQ ID NO: 53) and GeneRacer 3′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 2 minutes was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 2 minutes was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 2 minutes was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution, a primer PhF3dH-F8 (5′-AAGTTAATGGTGGAGGCAGTGATATGCTGA-3′; SEQ ID NO: 54) and GeneRacer 3′ Nested primer.
  • a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 2 minutes was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) and designated as PhF3′H 3′RACE/pCR4.
  • the 3′ DNA sequence of PhF3′H 3′RACE/pCR4 was determined (PhF3′H 3′RACE sequence).
  • 5′RACE was carried out by using a primer designed from the PhF3′H partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhF3dH-R6 (5′-CACTGCCTCCACCATTAACTTCGCCCTTCT-3′; SEQ ID NO: 55) and GeneRacer 5′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution, a primer PhF3dH-R5 (5′-CCTCCACCATTAACTTCGCCCTTCTCTATT-3′; SEQ ID NO: 56) and GeneRacer 5′ Nested primer.
  • a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4-TOPO (Invitrogen) and designated as PhF3′H 5′RACE/pCR4.
  • the 5′ DNA sequence of PhF3′H 5′RACE/pCR4 was determined (PhF3′H 5′RACE sequence).
  • PhF3′H gene was cloned on the basis of the PhF3′H 3′RACE sequence and the PhF3′H 5′RACE sequence.
  • RT-PCR was carried out by using the above-mentioned RNA, a random hexamer, a primer PhF3dH-F11E4 (5′-AAGAAGCAAATGGCATTCTTAACCTACCTG-3′; SEQ ID NO: 57) and a primer PhF3dH-R7G11 (5′-TTATCACTGCCTCCACCATTAACTTCGCCC-3′; SEQ ID NO: 58) and Ready-To-Go You Prime First Strand Beads (Amersham Biosciences). In the reaction, a step of 98° C.
  • PhF3′H/pCR4 The DNA sequence of the PhF3′H gene in PhF3′H/pCR4 was determined (PhF3′H; SEQ ID NO: 1), and the gene having the sequence from moth orchid was found to be novel. Homology analysis indicated that the amino acid sequence encoded by the DNA sequence has 59% homology to the amino acid sequence (GenBank accession No.: DQ787855) encoded by the F3′H gene from Sorghum bicolor .
  • PhF3′H/pCR4 was cut with EcoRI, and the cohesive end of DNA fragment from the plasmid was blunted with Klenow fragment. The DNA fragment was inserted at the SwaI cleavage site of pBS-P35T35, and clones were screened for the cDNA inserted in the sense direction to construct pBS-35S-PhF3′H.
  • pBS-35S-PhF3′H is DNA for expressing the moth orchid F3′H gene in plant cells.
  • DFR genes from various plants have been reported (GenBank accession Nos.: AAB62873, AAC17843, AAD49343, AAQ83576, AAU93766, AAY32600, AAY32601, AAY32602, BAB40789 and BAE79202), in the PCR, DFRD-F1 (5′-TTYCAYGTIGCIACNCCNATG-3′; SEQ ID NO: 59) and DFRD-R1 (5′-DATNGCRTCRTCRAACATYTC-3′; SEQ ID NO: 60) designed from the sequence of the above-mentioned DFR genes were used as the primers. In the reaction, a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer DFRD-F2 (5′-ATGAAYTTYCARWSIRARGAYCC-3′; SEQ ID NO: 61) and a primer DFRD-R2 (5′-RCAIATRTAICKNCIRTTNGC-3′; SEQ ID NO:62). In the reaction, a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 1 minute was repeated 40 cycles.
  • Nested PCR was carried out again by using the resulting reaction solution as the template, a primer DFRD-F3 (5′-GARAAYGARGTNATHAARCC-3′; SEQ ID NO: 63) and a primer DFRD-R3 (5′-RTCRTCIARRTGNACRAAYTG-3′; SEQ ID NO:64).
  • a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 30 seconds was repeated 40 cycles.
  • the resulting reaction product was cloned into pCR4/TOPO (Invitrogen) to obtain PhDFR-D/pCR4, and a partial DNA sequence of PhDFR-D/pCR4 was determined (PhDFR partial sequence).
  • 3′ RACE was carried out by using a primer designed from the PhDFR partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used for the PCR were PhDFR-F1 (5′-GGTCATGCAAAAGGTCGGGCAGCGTAA-3′; SEQ ID NO: 65) and GeneRacer 3′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer PhDFR-F2 (5′-GTGATCTTCACATCTTCCGCAGGAACAGT-3′; SEQ ID NO: 66) and GeneRacer 3′ Nested primer.
  • a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4/TOPO10 (Invitrogen) to obtain PhDFR3′RACE/pCR4, and the 3′ DNA sequence of PhDFR3′RACE/pCR4 was determined (PhDFR3′RACE sequence).
  • 5′RACE was carried out by using a primer designed from the PhDFR partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhDFR-R4 (5′-ATGATTCATTAAAAATCCGAAAAAAAGACCACTACAA-3′; SEQ ID NO: 67) and GeneRacer 5′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer PhDFR-R3 (5′-AACCATGCATAATAAAGCAGATGTGTAAAT-3′; SEQ ID NO: 68) and GeneRacer 5′ Nested primer.
  • a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
  • the resulting reaction product was cloned into pCR4/TOPO (Invitrogen) to obtain PhDFR 5′RACE/pCR4, and the 5′ DNA sequence of PhDFR 5′RACE/pCR4 was determined (PhDFR 5′RACE sequence).
  • RNA a primer PhDFR-F8A5 (5′-AAAAAATGGAGGATGTGAGGAAGGGTCCTGTT-3′; SEQ ID NO: 69), a primer PhDFR-R5 (5′-ACATGATTCATTAAAAATCCGAAAAAAAGACCA-3′; SEQ ID NO: 70) and Ready-To-Go You Prime First Strand Beads (Amersham Biosciences).
  • p35PhDFR DNA sequence of the full-length moth orchid DFR gene in p35PhDFR was determined (PhDFR; SEQ ID NO: 15), and the gene having the sequence from moth orchid was found to be novel. Homology analysis indicated that the amino acid sequence encoded by the DNA sequence has 86% homology to the amino acid sequence (GenBank accession No.: AF007096) encoded by the DFR gene of Bromheadia finlaysoniana .
  • p35PhDFR is DNA for expressing the moth orchid DFR gene in plant cells.
  • a primer ANS-dgF2 (5′-TICARGGBTAYGGIAGYARRYTIGCIRMYA-3′; SEQ ID NO: 71) and ANS-dgR2 (5′-GGYTCRCARAAIAYIRCCCAIGADA-3′; SEQ ID NO: 72) designed from a known ANS gene were used as the primers.
  • a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 40 cycles.
  • a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute was repeated 30 cycles.
  • the resulting reaction product was cloned into pCR4/TOPO (Invitrogen) to obtain PhANSfrag10, and a partial DNA sequence of PhANSfrag10 was determined (PhANS partial sequence).
  • 3′ RACE was carried out by using a primer designed from the PhANS partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR were PhANS3RACEGSP1 (5′-GCCCACACCGACGTCAGCTCCCTCTCCT-3′; SEQ ID NO: 73) and GeneRacer 3′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1.5 minutes was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 70° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer PhANS3RACEGSP2 (5′-CGTCGGGGATGCGCTCGAGATCCTCAGC-3′; SEQ ID NO: 74) and GeneRacer 3′ Nested primer.
  • a step of 94° C. for 10 seconds, 58° C. for 10 seconds and 72° C. for 1 minute was repeated 35 cycles.
  • the resulting reaction product was cloned into pCR4/TOPO (Invitrogen) to obtain PhANS3′RACE37, and the 3′ DNA sequence of PhANS3′RACE37 was determined (PhANS3′RACE sequence).
  • 5′RACE was carried out by using a primer designed from the PhANS partial sequence, the above-mentioned RNA and GeneRacer kit (Invitrogen).
  • the primers used in the PCR reaction were PhANS5RACEGSP1 (5′-AGTCCGCGGGTTCAGTCGGCCAGATGGT-3′; SEQ ID NO: 75) and GeneRacer 5′ primer.
  • a step of 94° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 5 cycles
  • a step of 94° C. for 30 seconds and 70° C. for 1.5 minutes was repeated 5 cycles
  • a step of 94° C. for 30 seconds, 70° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 25 cycles.
  • Nested PCR was carried out by using the resulting reaction solution as the template, a primer PhANS5RACEGSP2 (5′-CCGTCTTCTCCGGCGGGTAGACGAGGTG-3′; SEQ ID NO: 76) and GeneRacer 5′ Nested primer.
  • a step of 94° C. for 10 seconds, 58° C. for 10 seconds and 72° C. for 1 minute was repeated 35 cycles.
  • the resulting reaction product was cloned into pCR4/TOPO (Invitrogen) to obtain PhANS5′RACE15, and the 5′ DNA sequence of PhANS5′RACE15 was determined (PhANS 5′RACE sequence).
  • the full length of moth orchid ANS gene was cloned on the basis of the PhANS3′RACE sequence and the PhANS 5′RACE sequence.
  • PCR was carried out by using the above-mentioned cDNA, a primer PhANS init (5′-ATGGCCACCAAAGCAATCCCACC-3′; SEQ ID NO: 77), and a primer PhANS term (5′-TCAATCCACAGGCGCCTTCT-3′; SEQ ID NO: 78).
  • a step of 94° C. for 30 seconds, 69° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 35 cycles.
  • the resulting reaction product was cloned into the SwaI site of pBS-P35T35 to obtain p35PhANS1.
  • p35PhANS1 DNA sequence of the full-length ANS gene (PhANS1) in p35PhANS1 was determined (PhANS1; SEQ ID NO: 11), and the gene having the sequence from moth orchid was found to be novel. Homology analysis indicated that the amino acid sequence encoded by the DNA sequence has 58% homology to the amino acid sequence (GenBank accession No.: EF079869) encoded by the ANS gene of Anthurium.
  • p35PhANS1 is DNA for expressing the moth orchid ANS gene in plant cells.
  • RNA was isolated from petals of a bud of a commercially available Gerbera hybrid and used as the template to prepare cDNA by SuperscriptII First-Strand Synthesis System (Invitrogen). Then, RT-PCR was carried out by using this cDNA as the template.
  • GerF3H-F (5′-ATGACGCCTTTAACGCTCCT-3′; SEQ ID NO: 79) and GerF3H-R (5′-CTAGACCTTAGTCGTCTCATATACATG-3′; SEQ ID NO: 80) designed from a known Gerbera F3′H gene (GerF3′H) sequence (GenBank accession No.: Z17221) were used as the primers.
  • p35GerF3′H is DNA for expressing the Gerbera F3′H gene in plant cells.
  • RT-PCR was carried out by using the cDNA obtained in Example 4(1) as the template.
  • GerDFR-F (5′-ATGGAAGAGGATTCTCCGGC-3′; SEQ ID NO: 81) and GerDFR-R (5′-CTATTGGCCTTCTTTTGAACAACAAA-3′; SEQ ID NO: 82) designed from a known Gerbera DFR gene (GerDFR) sequence (GenBank accession No.: Z17221) were used as the primers.
  • the reaction was carried out by repeating a step of 98° C. for 10 seconds, 55° C. for 10 seconds and 72° C. for 1 minute and 30 seconds 45 cycles.
  • p35GerDFR is DNA for expressing the Gerbera DFR gene in plant cells.
  • RT-PCR was carried out by using the cDNA obtained in Example 4(1) as the template.
  • GerANS-F (5′-ATGGTGATTCAAGCAACCACA-3′; SEQ ID NO: 83) and GerANS-R (5′-CTAGTTTTGCATCACTTCGTCTTTAT-3′; SEQ ID NO: 84) designed from known Gerbera ANS gene (GerANS) sequence (GenBank accession No.: AY997842) were used as the primers.
  • a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute and 10 seconds was repeated 45 cycles.
  • p35GerANS is DNA for expressing the Gerbera ANS gene in plant cells.
  • RNA was isolated from petals of a bud of a commercially available Torenia ( Torenia tenteri ) and used as the template to prepare cDNA by SuperscriptII First-Strand Synthesis System (Invitrogen). Then, RT-PCR was carried out by using this cDNA as the template.
  • TorF3H1-F (5′-ATGAGTCCCTTAGCCTTGATGAT-3′; SEQ ID NO: 85) and TorF3H1-R (5′-TTAATAGACATGAGTGGCCAACC-3′; SEQ ID NO: 86) designed from a known Torenia F3′H gene (TorF3′H) sequence (GenBank accession AB057672) were used as the primers.
  • a step of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 1 minute and 10 seconds was repeated 45 cycles.
  • the resulting reaction product was cloned into the SwaI site of pBS-P35T35 to obtain a Torenia F3′H gene (p35TorF3′H) (SEQ ID NO: 87).
  • p35TorF3′H is DNA for expressing the Torenia F3′H gene in plant cells.
  • RT-PCR was carried out by using the cDNA obtained in Example 5(1) as the template.
  • oligonucleotides TorDFR-F (5′-ATGAGCATGGAAGTAGTAGTACCA-3′; SEQ ID NO: 89) and TorDFR-R (5′-CTATTCTATCTTATGTTCTCCATGG-3′; SEQ ID NO: 90) designed from a known Torenia DFR gene (TorDFR) sequence (GenBank accession AB012924) were used as the primers.
  • a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute and 10 seconds was repeated 45 cycles.
  • p35TorDFR is DNA for expressing the Torenia DFR gene in plant cells.
  • Petals of a white moth orchid ( Phal. amabilis ) were transfected with the following four gene sets by the method in Example 1, and the degrees of coloration of the petals were observed in accordance with Example 6 to identify the genes required to change the flower color.
  • Gerbera F3′H gene (GerF3′H)+ Gerbera DFR gene (GerDFR)+ Gerbera ANS gene (GerANS)
  • Petals of Phal. amabilis were transfected with the gene sets, and the coloration of cells in the petal treated with the above set were observed. Red cells appeared in the petals transfected with all the gene sets except (1) ( FIG. 3 ). The results indicate that in order to change petals to red, transfection with F3′H gene+DFR gene+ANS gene is insufficient, and transfection with at least four genes including F3H gene in addition to them is necessary.
  • Petals of a white moth orchid ( Phal. amabilis ) were transfected with the following three sets of genes each including a different F3′H gene, and the degrees of coloration were observed.
  • Torenia F3′H gene (TorF3′H)+moth orchid CHS gene (PhCHS3)+moth orchid CHI gene (PhCHI1)+moth orchid F3H gene (PhF3H1)+ Torenia DFR gene (TorDFR)+moth orchid ANS gene (PhANS1)
  • Petals of a white moth orchid ( Phal. amabilis ) were transfected with the following three sets of genes each including a different DFR gene, and the degrees of coloration were observed.
  • Gerbera DFR gene (GerDFR)+moth orchid CHS gene (PhCHS3)+moth orchid CHI gene (PhCHI1)+moth orchid F3H gene (PhF3H1)+ Gerbera F3′H gene (GerF3′H)+ Gerbera ANS gene (GerANS)
  • Torenia DFR gene (TorDFR)+moth orchid CHS gene (PhCHS3)+moth orchid CHI gene (PhCHI1)+moth orchid F3H gene (PhF3H1)+ Gerbera F3′H gene (GerF3′H)+ Gerbera ANS gene (GerANS)
  • Petals of a white moth orchid ( Phal. amabilis ) were transfected with the following two sets of genes each including a different ANS gene, and the degrees of coloration were observed.
  • Gerbera ANS gene GerANS+moth orchid CHS gene (PhCHS3)+moth orchid CHI gene (PhCHI1)+moth orchid F3H gene (PhF3H1)+ Gerbera F3′H gene (GerF3′H)+ Gerbera DFR gene (GerDFR)
  • the petals of a 1.7 cm-long bud of a commercial large pure white moth orchid were cotransfected with the CHS, CHI and F3H genes from a moth orchid (PhCHS3, PhCHI1 and PhF3H1) and the F3′H, DFR and ANS genes from Gerbera (GerF3′H, GerDFR and GerANS), and many red cells appeared in the petals.
  • Transformation of moth orchids can be carried out by Agrobacterium -mediated transformation, as well as microprojectile bombardment (Belamino and Mii, Plant Cell Reports (2000) 19:435-442, Mishiba et al., Plant Cell Reports (2005) 24: 297-303). Transforming DNAs for production of transgenic plants by these methods were constructed. Maps of these plasmids and the procedures for their preparation are shown in FIGS. 7 and 8 .
  • pBI-SAS1 is a plasmid which imparts hygromycin resistance to plants by Agrobacterium -mediated transformation.
  • FTcDNA was prepared from total RNA isolated from an Arabidopsis whole body by RT-PCR amplification.
  • AtFT 2nd-F (5′-GAAACCACCTGTTTGTTCAAGA-3′; SEQ ID NO: 91) and AtFT 2nd-R (5′-TCAATTGGTTATAAAGGAAGAAGC-3′; SEQ ID NO: 92) were used as the primers, and FT cDNA was inserted into the SwaI site of pBS-P35T35.
  • the clones carrying the cDNA insert in the sense direction were selected to construct pBS-P35S-FT.
  • transcription of FT is regulated by the CaMV 35S promoter and the CaMV 35S terminator.
  • Nucleotide substitutions were introduced into the two SphI sites of PhF3′H cDNA without amino acid substitution, and the resulting SphI-resistant cDNA was inserted into pBS-P35T35.
  • the nucleotide substitutions were carried out by PCR using PhF3dH-367F (5′-CGGTGCGCGGTGGCGTATGCTGAGGCGT-3′; SEQ ID NO: 93) and PhF3dH-394R (5′-ACGCCTCAGCATACGCCACCGCGCACCG-3′; SEQ ID NO: 94) as the primers and pBS-35S-PhF3′H as the template.
  • the PCR product was treated with Klenow fragment and cyclized.
  • PCR was carried out by using the resulting circular DNA as the template and PhF3dH-F11E4 (SEQ ID NO: 57) and PhF3dH-R7G12 (5′-CACCCTTTGCATAAATTTATGACATCAAGC-3′; SEQ ID NO: 95) as the primers.
  • the resulting PCR product was inserted into the SwaI cleavage site of pBS-P35T35, and the clones carrying the cDNA insert in the sense direction were selected to construct pBS-35S-mPhF3′H.
  • a nucleotide substitution was introduced into the SphI site of PhCHS3 cDNA without amino acid substitution to prepare a SphI-resistant cDNA.
  • the nucleotide substitution was carried out by DNA synthesis using PhCHS3-1038F (5′-GTAACATGTCGAGCGCTTGCGTTCTTTTCATACTCG-3′; SEQ ID NO: 96) and PhCHS3-1073R (5′-CGAGTATGAAAAGAACGCAAGCGCTCGACATGTTAC-3′; SEQ ID NO: 97) as the primers and p35PhCHS3 as the template and Pyrobest (Takara Bio Inc.). Then, the template plasmid was digested by DpnI treatment to construct pBS-35S-mPhCHS3.
  • p35PhCHI1 was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of p35PhF3H1 to construct pBS-35S-UP1, after p35PhF3H1 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-UP1 carries the cDNAs of PhCHI1 and PhF3H1 in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • p35GerANS was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of p35GerDFR to construct pBS-35S-Cya1 after p35GerDFR was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya1 carries the cDNAs of GerANS and GerDFR in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • p35GerF3′H was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-Cya1 to construct pBS-35S-Cya2 after pBS-35S-Cya1 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya2 carries the cDNAs of GerF3′H, GerANS and GerDFR in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya2 was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-UP1 to construct pBS-35S-Cya3 after pBS-35S-UP1 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya3 carries the cDNAs of GerF3′H, GerANS, GerDFR, PhCHI and PhF3H in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya3 was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-mPhCHS3 to construct pBS-35S-Cya4 after pBS-35S-mPhCHS3 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya4 carries the cDNAs of GerF3′H, GerANS, GerDFR, PhCHI, PhF3H and mPhCHS3 in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-mPhF3′H was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-FT to construct pBS-35S-Cya10 after pBS-35S-FT was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya10 carries the cDNAs of mPhF3′H and FT in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • p35 TorDFR was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of p35PhANS1 to construct pBS-35S-DeI16 after p35PhANS1 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-DeI16 carries the cDNAs of TorDFR and PhANS1 in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Dell 6 was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-UP1 to construct pBS-35S-UP4 after pBS-35S-UP1 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-UP4 carries the cDNAs of TorDFR, PhANS1, PhCHI1 and PhF3H1 in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-mPhF3′H was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-UP4 to construct pBS-35S-Cya11 after pBS-35S-UP4 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya11 carries the cDNAs of mPhF3′H, TorDFR, PhANS, PhCHI and PhF3H in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya10 was cut with AscI, then blunted with Klenow fragment and further cut with SphI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted into the SphI cleavage site of pBS-35S-UP4 to construct pBS-35S-Cya12 after pBS-35S-UP4 was cut with XbaI and blunted with Klenow fragment.
  • the plasmid pBS-35S-Cya12 carries the cDNAs of mPhF3′H, FT, TorDFR, PhANS, PhCHI and PhF3H in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya3 was cut with SphI, then blunted with Klenow fragment and further cut with AscI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted between the AscI and SwaI cleavage sites of pBI-SAS1 to construct pBIH-35S-Cya3.
  • pBIH-35S-Cya3 is a binary vector with a T-DNA region carrying a selectable marker HPT and the cDNAs of GerF3′H, GerANS, GerDFR, PhCHI and PhF3H in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya4 was cut with SphI, then blunted with Klenow fragment and further cut with AscI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted between the AscI and SwaI cleavage sites of pBI-SAS1 to construct pBIH-35S-Cya4.
  • pBIH-35S-Cya4 is a binary vector with a T-DNA region carrying a selectable marker HPT and the cDNAs of GerF3′H, GerANS, GerDFR, PhCHI, PhF3H and mPhCHS3 in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya11 was cut with SphI, then blunted with Klenow fragment and further cut with AscI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted between the AscI and SwaI cleavage sites of pBI-SAS1 to construct pBIH-35S-Cya11.
  • pBIH-35S-Cya11 is a binary vector with a T-DNA region carrying a selectable marker HPT and the cDNAs of mPhF3′H, TorDFR, PhANS, PhCHI and PhF3H in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • pBS-35S-Cya12 was cut with SphI, then blunted with Klenow fragment and further cut with AscI to cut out a DNA fragment from the plasmid.
  • the DNA fragment was inserted between the AscI and SwaI cleavage sites of pBI-SAS1 to construct pBIH-35S-Cya12.
  • pBIH-35S-Cya12 is a binary vector with a T-DNA region carrying a selectable marker HPT and the cDNAs of mPhF3′H, FT, TorDFR, PhANS, PhCHI and PhF3H in this order under the control of the CaMV 35S promoter and the CaMV 35S terminator.
  • a moth orchid was transfected with the binary vector DNAs constructed in Example 12 (pBIH-35S-Cya3, pBIH-35S-Cya4, pBIH-35S-Cya11 and pBIH-35S-Cya12) and Agrobacterium EHA101 strain, and transgenic moth orchids were selected in the presence of 50 mg/ml hygromycin to obtain moth orchids having the genes recited in Example 12 integrated into the chromosomes.
  • the resulting transgenic moth orchids can clonally propagate through induction of PLBs from a part of the plant such as axillary buds of flower stalks.
  • the transgenic moth orchids can be used as a parent in cross breeding to obtain a progeny having the introduced genes.
  • the present invention makes it possible to produce a new variety of moth orchid having a pink or red flower color from a white moth orchid by changing the flower color while maintaining the superiorities of other characters than flower color and is industrially applicable to a wide variety of use.

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CN102660561A (zh) * 2012-05-25 2012-09-12 西南大学 桑树花青素生物合成三个关键基因及应用
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